Zebrafish and Drug Development: A Behavioral Assay System for Probing Nicotine Function in Larval Zebrafish

  • Henning SchneiderEmail author
  • Eric W. Klee
  • Karl J. Clark
  • Andrew M. Petzold
  • Vanessa L. Mock
  • Julia M. Abarr
  • Jennifer L. Behrens
  • Ryan E. Edelen
  • Bryan A. Edwards
  • Joshua S. Hobgood
  • Meghann E. Pogue
  • Nishant K. Singh
  • Stephen C. Ekker
Part of the Neuromethods book series (NM, volume 66)


The attributes of the zebrafish (Danio rerio) make it an excellent model system for the development and discovery of new drugs. A robust behavioral assay is described that has been used successfully in studies of nicotine biology. The movement response of a group of larval zebrafish is measured over a 5-min period following application of nicotine. Pretreatment of larvae is employed to identify chemical compounds that reduce locomotor responses to acute nicotine. Activity plots provide an assessment of the biological activity and specificity of neuroactive chemical compounds in intact organisms. The experimental setup can be established in a research or teaching laboratory. The described behavioral assay can be used in pharmacological studies for the characterization of new chemical compounds and is a powerful tool for the discovery of behavioral zebrafish mutants.

Key words

Behavioral testing Drug discovery Locomotion Nicotine Serotonin Zebrafish 



Support for this project was provided by DePauw University, NIH DA014546 to SCE, and Mayo Foundation. We thank the team of the Mayo Clinic Zebrafish facility and members of the Ekker-lab for their help and suggestions.


  1. 1.
    Kenakin T (2003) Predicting therapeutic value in the lead optimization phase of drug discovery. Nat Rev Drug Discov 2:429–438PubMedCrossRefGoogle Scholar
  2. 2.
    Tecott LH, Nestler EJ (2004) Neurobehavioral assessment in the information age. Nat Neurosci 7:462–466PubMedCrossRefGoogle Scholar
  3. 3.
    Kenakin TP (2009) Cellular assays as portals to seven-transmembrane receptor-based drug discovery. Nat Rev Drug Discov 8:617–626PubMedCrossRefGoogle Scholar
  4. 4.
    Baldessari D, Mione M (2008) How to create the vascular tree? (Latest) help from the zebrafish. Pharmacol Ther 118:206–230PubMedCrossRefGoogle Scholar
  5. 5.
    Berghmans S, Hunt J, Roach A, Goldsmith P (2007) Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvulsants. Epilepsy Res 75:18–28PubMedCrossRefGoogle Scholar
  6. 6.
    Buckley CE, Marguerie A, Roach AG, Goldsmith P, Fleming A, Alderton WK, Franklin RJM (2010) Drug reprofiling using zebrafish identifies novel compounds with potential pro-myelination effects. Neuropharmacology 59:149–159PubMedCrossRefGoogle Scholar
  7. 7.
    Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, Elegante MF, Elkhayat SI, Bartels BK, Tien AK, Tien DH, Mohnot S, Beeson E, Glasgow E, Amri H, Zukowska Z, Kalueff AV (2009) Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Res 205:38–44PubMedCrossRefGoogle Scholar
  8. 8.
    Kitambi SS, McCulloch KJ, Peterson RT, Malicki JJ (2009) Small molecule screen for compounds that affect vascular development in the zebrafish retina. Mech Dev 126:464–477PubMedCrossRefGoogle Scholar
  9. 9.
    Love DR, Pichler FB, Dodd A, Copp BR, Greenwood DR (2004) Technology for high-throughput screens: the present and future using zebrafish. Curr Opin Biotechnol 15:564–571PubMedCrossRefGoogle Scholar
  10. 10.
    MacRae CA, Peterson RT (2003) Zebrafish-based small molecule discovery. Chem Biol 10:901–908PubMedCrossRefGoogle Scholar
  11. 11.
    McAleer MF, Davidson C, Davidson WR, Yentzer B, Farber SA, Rodeck U, Dicker AP (2005) Novel use of zebrafish as a vertebrate model to screen radiation protectors and sensitizers. Int J Radiat Oncol Biol Phys 61:10–13PubMedCrossRefGoogle Scholar
  12. 12.
    McGrath P, Li C-Q (2008) Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov Today 13:394–401PubMedCrossRefGoogle Scholar
  13. 13.
    McKinley ET, Baranowski TC, Blavo DO, Cato C, Doan TN, Rubinstein AL (2005) Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons. Mol Brain Res 141:128–137PubMedCrossRefGoogle Scholar
  14. 14.
    Meeker ND, Trede NS (2008) Immunology and zebrafish: spawning new models of human disease. Dev Comp Immunol 32:745–757PubMedCrossRefGoogle Scholar
  15. 15.
    Murphey RD, Zon LI (2006) Small molecule screening in the zebrafish. Methods 39:255–261PubMedCrossRefGoogle Scholar
  16. 16.
    Nguyen CT, Lu Q, Wang Y, Chen J-N (2008) Zebrafish as a model for cardiovascular development and disease. Drug Discov Today Dis Models 5:135–140PubMedCrossRefGoogle Scholar
  17. 17.
    Peterson RT (2004) Discovery of therapeutic targets by phenotype-based zebrafish screens. Drug Discov Today Technol 1:49–54CrossRefGoogle Scholar
  18. 18.
    Peterson RT, Fishman MC (2004) Discovery and use of small molecules for probing biological processes in zebrafish. Methods Cell Biol 76:569–591PubMedCrossRefGoogle Scholar
  19. 19.
    Redfern WS, Waldron G, Winter MJ, Butler P, Holbrook M, Wallis R, Valentin J-P (2008) Zebrafish assays as early safety pharmacology screens: paradigm shift or red herring? J Pharmacol Toxicol Methods 58:110–117PubMedCrossRefGoogle Scholar
  20. 20.
    Sumanas S, Lin S (2004) Zebrafish as a model system for drug target screening and validation. Drug Discov Today Targets 3:89–96CrossRefGoogle Scholar
  21. 21.
    Winter MJ, Redfern W, Hayfield A, Owen S, Valentin JP, Hutchinson T (2008) Zebrafish embryo-larval locomotion as a frontloaded screen for assessing seizure liability during early drug discovery. J Pharmacol Toxicol Methods 58:169CrossRefGoogle Scholar
  22. 22.
    Eddins D, Petro A, Williams P, Cerutti DT, Levin ED (2009) Nicotine effects on learning in zebrafish: the role of dopaminergic systems. Psychopharmacology (Berl) 202:103–109CrossRefGoogle Scholar
  23. 23.
    Bencan Z, Levin ED (2008) The role of alpha7 and alpha4 beta2 nicotinic receptors in the nicotine-induced anxiolytic effect in zebrafish. Physiol Behav 95:408–412PubMedCrossRefGoogle Scholar
  24. 24.
    Levin ED, Bencan Z, Cerutti DT (2007) Anxiolytic effects of nicotine in zebrafish. Physiol Behav 90:54–58PubMedCrossRefGoogle Scholar
  25. 25.
    Levin ED, Chen E (2004) Nicotinic involvement in memory function in zebrafish. Neurotoxicol Teratol 26:731–735PubMedCrossRefGoogle Scholar
  26. 26.
    Klee EW, Ebbert JO, Schneider H, Hurt RD, Ekker SC (2011) Zebrafish for the study of the biological effects of nicotine. Nicotine Tob Res 13:301–312PubMedCrossRefGoogle Scholar
  27. 27.
    Petzold AM, Balciunas D, Sivasubbu S, Clark KJ, Bedell VM, Westcot SE, Myers SR, Moulder GL, Thomas MJ, Ekker SC (2009) Nicotine response genetics in the zebrafish. Proc Natl Acad Sci U S A 106:18662–18667PubMedCrossRefGoogle Scholar
  28. 28.
    Drapeau P, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Brustein E (2002) Development of the locomotor network in zebrafish. Prog Neurobiol 68:85–111PubMedCrossRefGoogle Scholar
  29. 29.
    Buss RR, Drapeau P (2001) Synaptic drive to motoneurons during fictive swimming in the developing zebrafish. J Neurophysiol 86:197–210PubMedGoogle Scholar
  30. 30.
    Budick SA, O’Malley DM (2000) Locomotor repertoire of the larval zebrafish: swimming, turning and prey capture. J Exp Biol 203:2565–2579PubMedGoogle Scholar
  31. 31.
    Thirumalai V, Cline HT (2008) Endogenous dopamine suppresses initiation of swimming in prefeeding zebrafish larvae. J Neurophysiol 100:1635–1648PubMedCrossRefGoogle Scholar
  32. 32.
    Lawrence C (2007) The husbandry of zebrafish (Danio rerio): a review. Aquaculture 269:1–20CrossRefGoogle Scholar
  33. 33.
    MacPhail RC, Brooks J, Hunter DL, Padnos B, Irons TD, Padilla S (2009) Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Neurotoxicology 30:52–58PubMedCrossRefGoogle Scholar
  34. 34.
    Sallinen V, Sundvik M, Reenila I, Peitsaro N, Khrustalyov D, Anichtchik O, Toleikyte G, Kaslin J, Panula P (2009) Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish. J Neurochem 109:403–415PubMedCrossRefGoogle Scholar
  35. 35.
    Emran F, Rihel J, Dowling JE (2008) A behavioral assay to measure responsiveness of zebrafish to changes in light intensities. J Vis Exp 3(20):923. doi: 10.3791/923 Google Scholar
  36. 36.
    Budick SB, O’Malley DM (2000) Minimal behavioural deficits are observed after laser-ablation of the nMLF in larval zebrafish. Am Zool 40:959Google Scholar
  37. 37.
    Borla MA, Palecek B, Budick S, O’Malley DM (2002) Prey capture by larval zebrafish: evidence for fine axial motor control. Brain Behav Evol 60:207–229PubMedCrossRefGoogle Scholar
  38. 38.
    McLean DL, Fetcho JR (2011) Movement, technology and discovery in the zebrafish. Curr Opin Neurobiol 21:110–115PubMedCrossRefGoogle Scholar
  39. 39.
    Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, Haggarty SJ, Kokel D, Rubin LL, Peterson RT, Schier AF (2010) Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science 327:348–351PubMedCrossRefGoogle Scholar
  40. 40.
    Kokel D, Bryan J, Laggner C, White R, Cheung CY, Mateus R, Healey D, Kim S, Werdich AA, Haggarty SJ, Macrae CA, Shoichet B, Peterson RT (2010) Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nat Chem Biol 6:231–237PubMedCrossRefGoogle Scholar
  41. 41.
    Pardo-Martin C, Chang TY, Koo BK, Gilleland CL, Wasserman SC, Yanik MF (2010) High-throughput in vivo vertebrate screening. Nat Methods 7:634–636PubMedCrossRefGoogle Scholar
  42. 42.
    Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310PubMedCrossRefGoogle Scholar
  43. 43.
    Brand M, Granato M, Nüsslein-Volhard C (2002) Keeping and raising zebrafish. In: Nüsslein-Volhard C, Dahm R (eds) Zebrafish, 1st edn. Oxford University Press, New York, NY, pp 7–37Google Scholar
  44. 44.
    Burgess HA, Granato M (2007) Modulation of locomotor activity in larval zebrafish during light adaptation. J Exp Biol 210:2526–2539PubMedCrossRefGoogle Scholar
  45. 45.
    Murphey RD, Stern HM, Straub CT, Zon LI (2006) A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chem Biol Drug Des 68:213–219PubMedCrossRefGoogle Scholar
  46. 46.
    Usenko CY, Harper SL, Tanguay RL (2007) In vivo evaluation of carbon fullerene toxicity using embryonic zebrafish. Carbon N Y 45:1891–1898PubMedCrossRefGoogle Scholar
  47. 47.
    Clark KJ, Boczek NJ, Ekker SC (2011) Stressing zebrafish for behavioral genetics. Rev Neurosci 22:49–62PubMedGoogle Scholar
  48. 48.
    Sackerman J, Donegan JJ, Cunningham CS, Nguyen NN, Lawless K, Long A, Benno RH, Gould GG (2010) Zebrafish behavior in novel environments: effects of acute exposure to anxiolytic compounds and choice of Danio rerio line. Int J Comp Psychol 23:43–61PubMedGoogle Scholar
  49. 49.
    Hicks C, Sorocco D, Levin M (2006) Automated analysis of behavior: a computer-controlled system for drug screening and the investigation of learning. J Neurobiol 66:977–990PubMedCrossRefGoogle Scholar
  50. 50.
    Branson K, Robie AA, Bender J, Perona P, Dickinson MH (2009) High-throughput ethomics in large groups of Drosophila. Nat Methods 6:451–457PubMedCrossRefGoogle Scholar
  51. 51.
    Dews PB (1955) Studies on behavior. II. The effects of pentobarbital, methamphetamine and scopolamine on performances in pigeons involving discriminations. J Pharmacol Exp Ther 115:380–389PubMedGoogle Scholar
  52. 52.
    Dews PB (1955) Studies on behavior. I. Differential sensitivity to pentobarbital of pecking performance in pigeons depending on the schedule of reward. J Pharmacol Exp Ther 113:393–401PubMedGoogle Scholar
  53. 53.
    Clark KJ, Balciunas D, Pogoda HM, Ding Y, Westcot SE, Bedell VM, Greenwood TM, Urban MD, Skuster KJ, Petzold AM, Ni J, Nielsen AL, Patowary A, Scaria V, Sivasubbu S, Xu X, Hammerschmidt M, Ekker SC (2011) In vivo protein trapping produces a functional expression codex of the vertebrate proteome. Nat Methods 8:506–512PubMedCrossRefGoogle Scholar
  54. 54.
    Ekker SC (2000) Morphants: a new systematic vertebrate functional genomics approach. Yeast 17:302–306PubMedCrossRefGoogle Scholar
  55. 55.
    Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC (2009) A primer for morpholino use in zebrafish. Zebrafish 6:69–77PubMedCrossRefGoogle Scholar
  56. 56.
    Nasevicius A, Ekker SC (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26:216–220PubMedCrossRefGoogle Scholar
  57. 57.
    Moens CB, Donn TM, Wolf-Saxon ER, Ma TP (2008) Reverse genetics in zebrafish by TILLING. Brief Funct Genomic Proteomic 7:454–459PubMedCrossRefGoogle Scholar
  58. 58.
    Wang D, Jao LE, Zheng N, Dolan K, Ivey J, Zonies S, Wu X, Wu K, Yang H, Meng Q, Zhu Z, Zhang B, Lin S, Burgess SM (2007) Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. Proc Natl Acad Sci U S A 104:12428–12433PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Henning Schneider
    • 1
    Email author
  • Eric W. Klee
    • 2
  • Karl J. Clark
    • 2
  • Andrew M. Petzold
    • 3
  • Vanessa L. Mock
    • 1
  • Julia M. Abarr
    • 1
  • Jennifer L. Behrens
    • 1
  • Ryan E. Edelen
    • 1
  • Bryan A. Edwards
    • 1
  • Joshua S. Hobgood
    • 1
  • Meghann E. Pogue
    • 1
  • Nishant K. Singh
    • 1
  • Stephen C. Ekker
    • 2
  1. 1.Department of BiologyDePauw UniversityGreencastleUSA
  2. 2.Mayo Addiction Research Center and Department of Biochemistry and Molecular BiologyMayo ClinicRochesterUSA
  3. 3.Center for Learning InnovationUniversity of Minnesota RochesterRochesterUSA

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